Since the first successful human pregnancy by way of in vitro fertilization (IVF) was achieved in 1978, ART has helped thousands of thousands of women to overcome infertility problem
Normally, during the follicular phase of a woman's reproductive cycle, only a single follicle grows to the preovulatory stage and releases its oocyte for potential fertilization. However, current IVF treatment requires that multiple oocytes be harvested. Therefore, women are normally pre-treated for approximately two or three weeks with gonadotropin releasing hormone agonists (GnRHa). After pituitary suppression has been achieved, human menopausal gonadotropin (HMG) or purified follicle-stimulating hormone (FSH) is administered to induce development of multiple follicles. Once two leading follicles have reached a diameter of at least about 18-20 mm, the patient is typically given 5,000 to 10,000 IU of human chorionic gonadotropin (HCG) to trigger final oocyte maturation. Approximately 36 hours later, a large number of oocytes (10 on average) are collected.
The harvested oocyte is then fertilized with the sperm. In many instances of human infertility, fertilization is effected by intracytoplasmic sperm injection (ICSI). Successful IVF generally requires that the oocyte have reached the metaphase-II (M-II) stage before fertilization is attempted. A M-II oocyte is ready to accept the sperm and be fertilized. Metaphase-II is characterized by exclusion of one polar body from the cytoplasm, and is typically detected by visual identification under a microscope.
The harvested oocyte remains surrounded by cumulus cells that support and promote the final maturation and development of the oocyte. The cumulus cells interfere with ICSI and obscure microscopic observation of the oocyte. Therefore, prior to ICSI, it is necessary to denude the oocyte of cumulus cells to permit determination of the level of oocyte maturity. This is generally accomplished by enzymatic and mechanical stripping of the cumulus cells.
Unfortunately, not all of the oocytes have matured to the M-II stage when they are harvested in ovarian stimulation. As a result of follicular asynchrony, in general, at least 10-15% of oocytes are still immature, and are at the germinal vesicle (GV) or metaphase-I (M-I) stage, at the time of harvest. These immature oocytes, denuded of cumulus cells, are discarded at most IVF clinics.
It would be desirable if these immature GV and M-I stage oocytes, presently wasted, could be brought to maturity, in vitro, and then successfully fertilized. Moreover, it would be desirable to be able to harvest immature oocytes from unstimulated ovaries and mature them to the M-II stage by way of in vitro maturation (IVM), thereby avoiding the entire regimen of treatment of women with GnRHa and stimulation of ovaries with gonadotropin. These procedures of ovarian stimulation require frequent blood sampling and ultrasound monitoring, at considerable cost. Moreover, current IVF treatment causes substantial discomfort and, in some cases, results in ovarian hyperstimulation syndrome (OHSS) and/or even death. There is also anxiety that the long-term effects of repeated ovarian stimulation may increase the risk of ovarian, endometrial and breast cancer.
Thus, there is substantial interest in developing IVF techniques that rely upon in vitro maturation of oocytes, rather than harvesting mature oocytes by ovarian stimulation. But only a few live births have been obtained from cumulus-denuded oocytes matured in vitro (Nagy et al., 1996; Jaroudi et al., 1997; Edirisinghe et al., 1997). Clearly then, there is an opportunity for improving the maturational and developmental competence of cumulus-denuded immature oocytes matured in vitro.
Recovery of immature oocytes followed by IVM of these immature oocytes is a potentially useful treatment for women with polycystic ovarian syndrome (PCOS) related infertility. PCOS is one of the most common reproductive disorders in women of childbearing age. It has a heterogeneous presentation, which is clinically characterized by anovulation and hyperandrogenism, and on pelvic ultrasound examination shows numerous antral follicles within the ovaries (Adams et al., 1986). There is a significantly higher risk of OHSS in these group of women compared with normal ovaries (MacDougall et al., 1993). Although the first pregnancy and live birth from immature oocytes retrieved from women with PCOS followed by IVM has been reported, the success rates are still low because oocyte maturation rates are relatively low.
Some attempts at developing IVM media and methods have involved the use of human follicular fluid, in an effort to mimic the conditions within the follicle. In view of the risk of transmission of disease, allergic reaction, etc., use of biological fluids in IVM techniques is prohibited in most countries. Instead, the use of a “chemically-defined medium”, wherein the chemical composition of all of the ingredients is known, is preferred.
Mammalian immature oocytes removed from antral follicles will resume meiosis spontaneously without hormonal stimulation when cultured in a simple medium (Pincus & Enamann, 1935). However, oocyte maturation in vitro is profoundly affected by culture conditions.
Most attempts at human IVM have employed tissue culture medium 199 (TCM-199) as the maturation medium for immature oocytes (Trounson et al., 1994; Barnes et al., 1995; Chian et al., 1999a, 1999b, 2000, 2001). TCM-199 is a complex medium that contains many components, and was designed for in vitro culture of somatic cells, not germ cells. Notably, TCM-199 does not contain any growth factors. TCM-199 was first used for sheep oocyte IVM (Moor & Trounson, 1977), and has since been used routinely for bovine oocyte IVM (Brackett & Zuelke, 1993).
Although different culture media have been used to mature human oocytes (Shea et al., 1975; Cha et al., 1991; Trounson et al., 1994; Cha & Chian, 1998; Chian et al., 1999a, 1999b, 2000), it has been indicated recently that there is no apparent benefit for oocyte maturation, fertilization and embryonic development from use of these culture media (Trounson et al., 1998; 2001).
Thus, there remains a need for methods for maturing human oocytes in vitro.